Quantifying interleaflet coupling of phase behavior and observing anti-registered phases in asymmetric lipid bilayers

Model asymmetric lipid bilayers provide a powerful platform for probing how lateral phase behavior in one leaflet is coupled to that of the opposing leaflet. Here, we use calcium-induced hemifusion to generate asymmetric giant unilamellar vesicles (aGUVs) and investigate how lipid composition modulates interleaflet coupling of liquid-liquid phase separation. Symmetric GUVs composed of cholesterol, the high-melting lipid DPPC, and a low-melting phosphatidylcholine (either 14:1-PC or 16:1-PC) were prepared at compositions exhibiting coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases. Hemifusion with a uniformly mixed supported lipid bilayer composed of the low-melting lipid and cholesterol selectively altered the outer leaflet composition, producing aGUVs with controlled but variable asymmetry. Quantification of outer leaflet exchange using both probe-exit and probe-entry fluorescence measurements revealed substantial vesicle-to-vesicle variability within a given preparation, resulting in overlapping populations of phase-separated and uniformly mixed aGUVs. To account for this variability, we developed a population-based, coupled-distributions framework that enables robust determination of the asymmetric miscibility boundary, defined as the outer leaflet composition at which macroscopic phase separation is suppressed. Independent analyses of probe-exit and probe-entry data yielded consistent boundary locations. Comparing the two lipid systems, we find that aGUVs containing 14:1-PC require significantly greater outer leaflet exchange to abolish phase separation than those containing 16:1-PC. Only in the 14:1-PC system do we observe vesicles exhibiting coexistence of distinct anti-registered phases, a theoretically predicted but rarely observed regime consistent with large hydrophobic mismatch. By expressing both symmetric and asymmetric miscibility boundaries in a common fractional-coordinate framework, we introduce a phenomenological parameter, ${\Delta }^{*}$ , that quantifies the direction and strength of interleaflet coupling of phase behavior. Together, these results demonstrate that modest changes in lipid chain length can markedly alter asymmetric miscibility boundaries and provide a quantitative link between experimental observations, leaflet dominance concepts, and coupled-leaflet theories of membrane organization.